Paroxysmal Cold Hemoglobinuria: Diagnosis From the Blood Smear.

Phagocytosis of red cells by neutrophils, referred to as neutrophil erythrophagocytosis, on the blood smear as a helpful diagnostic sign of paroxysmal cold hemoglobinuria is underrecognized. We present a child with paroxysmal cold hemoglobinuria and prominent neutrophil erythrophagocytosis to highlight the importance of this finding in the diagnosis of paroxysmal cold hemoglobinuria.

Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias.

Mutations in more than 70 genes cause hereditary anemias (HA), a highly heterogeneous group of rare/low frequency disorders in which we included: hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA) and Diamond-Blackfan anemia; hemolytic anemias due to erythrocyte membrane defects, as hereditary spherocytosis and stomatocytosis; hemolytic anemias due to enzymatic defects. The study describes the diagnostic workflow for HA, based on the development of two consecutive versions of a targeted-NGS panel, including 34 and 71 genes, respectively. Seventy-four probands from 62 unrelated families were investigated. Our study includes the most comprehensive gene set for these anemias and the largest cohort of patients described so far. We obtained an overall diagnostic yield of 64.9%. Despite 54.2% of cases showed conclusive diagnosis fitting well to the clinical suspicion, the multi-gene analysis modified the original clinical diagnosis in 45.8% of patients (nonmatched phenotype-genotype). Of note, 81.8% of nonmatched patients were clinically suspected to suffer from CDA. Particularly, 45.5% of the probands originally classified as CDA exhibited a conclusive diagnosis of chronic anemia due to enzymatic defects, mainly due to mutations in PKLR gene. Interestingly, we also identified a syndromic CDA patient with mild anemia and epilepsy, showing a homozygous mutation in CAD gene, recently associated to early infantile epileptic encephalopathy-50 and CDA-like anemia. Finally, we described a patient showing marked iron overload due to the coinheritance of PIEZO1 and SEC23B mutations, demonstrating that the multi-gene approach is valuable not only for achieving a correct and definitive diagnosis, but also for guiding treatment.

Diagnosis and treatment of MYH9-RD in an Australasian cohort with thrombocytopenia.

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis.

Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.

Normal paediatric bone marrow: magnetic resonance imaging appearances from birth to 5 years.

INTRODUCTION: Bone marrow (BM) assessment is an important aspect of paediatric MRI, with the marrow cavity visible on almost every clinical MR examination. In practice, however, assessment for marrow infiltration in paediatric patients can be challenging. Our aim was to review the MRI appearance of normal BM from 0 to 5 years. METHODS: Consecutive body MR examinations over 7 years were retrospectively reviewed in patients aged 0-5 years. Patients with anticipated BM abnormality were excluded. All patients had imaging of the spine and/or pelvis with T1-weighted (T1) ± T2 with fat saturation, post-contrast T1-weighted, diffusion-weighted or out-of-phase sequences. RESULTS: Nineteen patients were included: nine (47%) infants, 11 (58%) boys, mean age 18.2 months (range 1 day to 3 years and 10 months). On T1 imaging, 69% infant marrow sites and 24% in children >1 year were isointense, the remainder were hyperintense. One hundred per cent BM was T2 fat saturation hyperintense. Enhancement following contrast was seen in 16% of BM sites. Restricted diffusion was seen in 100% infant BM and 50% BM in children >1 year. On out-of-phase imaging, no signal loss was seen in infants, and 21% BM in children >1 year showed signal loss. CONCLUSION: Due to normal age-related differences in BM histology, MRI for marrow assessment in infants and young children can be misleading and may mimic marrow infiltration.

Kaolin clotting time.

The kaolin clotting time (KCT) is a sensitive test used in the laboratory detection of lupus anticoagulants (LA) (Derksen and de Groot, Thromb Res 114:521-526, 2004). It is essentially an activated partial thromboplastin time (APTT) test with no added phospholipid. Kaolin acts as the activator in the KCT. In the absence of additional phospholipid reagent, the quality of the test sample is extremely important since the generation of thrombin completely depends on the presence of residual cell membranes and plasma lipids (Derksen and de Groot, Thromb Res 114:521-526, 2004). Since the test contains no exogenous phospholipid, a confirmatory test using excess phospholipid is required to confirm the presence of lupus anticoagulant in the sample (Court, Br J Biomed Sci 54:287-298, 1997).

The dilute Russell's viper venom time.

The dilute Russell's viper venom time is a clot-based test used in the detection of the lupus anticoagulant in the laboratory. Lupus anticoagulants and the overall approach for their detection are described in Chapter 7.

Platelet neutralization test.

The platelet neutralization test is used in the laboratorvy diagnosis of the lupus anticoagulant (Laffan and Manning, Dacie and Lewis practical haematology. Churchill Livingstone, Philadelphia, PA, pp 445-446, 2006). The lupus anticoagulant typically causes prolongation of phospholipid-dependent coagulation tests such as APTT, DRVVT, etc. The phospholipid-dependent nature of the LA can be demonstrated by adding washed and "fractured" platelets as a source of phospholipid and repeating the tests. If an LA is present in the sample, the addition of platelets will correct the prolonged clotting times. This appears to be due to the ability of the platelets to absorb the LA and negate its effect on the clotting time (Br J Haematol 109:704-715, 2001).